Publications by authors named "Ellen Heitzer"

101 Publications

Validation of a next-generation sequencing assay for the detection of EGFR mutations in cell-free circulating tumor DNA.

Exp Mol Pathol 2021 Sep 21;123:104685. Epub 2021 Sep 21.

Institute of Human Genetics, Diagnostic and Research Center for Molecular BioMedicine, Medical University of Graz, Graz, Austria. Electronic address:

Detection of EGFR mutations from blood plasma represents a gentle, non-invasive alternative to rebiopsy and can therefore be used for therapy monitoring of non-small-cell lung cancer (NSCLC) patients. The aim of this project was to investigate whether the Reveal ctDNA™ 28 NGS assay (ArcherDX, Boulder, CO), has a comparable sensitivity and specificity to droplet digital PCR (ddPCR, gold-standard) and is therefore suitable for therapy monitoring of progressing lung cancer patients. First, we validated the NGS assay with a commercially available reference material (SeraCare, Massachusetts, US). Using an input of 22 ng, a sensitivity of 96% and a specificity of 100% could be achieved for variant allele frequencies (VAF) of 0.5%. For variants at a VAF of 0.1% the sensitivity was substantially reduced. Next, 28 plasma samples from 16 patients were analyzed and results were compared to existing ddPCR data. This comparative analysis of patient samples revealed a concordance of 91% between NGS and ddPCR. These results confirm that the Reveal ctDNA™ 28 NGS assay can be used for therapy monitoring of patients under TKI therapy. However, due to the slightly superior sensitivity of ddPCR, a combination of NGS (with broad coverage of a large number of genomic loci) and ddPCR (with targeted highly sensitive detection of specific mutations) might be the ideal approach.
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http://dx.doi.org/10.1016/j.yexmp.2021.104685DOI Listing
September 2021

Molecular profiling of soft-tissue sarcomas with FoundationOne Heme identifies potential targets for sarcoma therapy: a single-centre experience.

Ther Adv Med Oncol 2021 25;13:17588359211029125. Epub 2021 Jul 25.

Division of Clinical Oncology, Medical University of Graz, Graz, Austria.

Background: Molecular diagnosis has become an established tool in the characterisation of adult soft-tissue sarcomas (STS). FoundationOne Heme analyses somatic gene alterations in sarcomas DNA and RNA-hotspot sequencing of tumour-associated genes.

Methods: We evaluated FoundationOne Heme testing in 81 localised STS including 35 translocation-associated and 46 complex-karyotyped cases from a single institution.

Results: Although FoundationOne Heme achieved broad patient coverage and identified at least five genetic alterations in each sample, the sensitivity for fusion detection was rather low, at 42.4%. Nevertheless, potential targets for STS treatment were detected using the FoundationOne Heme assay: complex-karyotyped sarcomas frequently displayed copy-number alterations of common tumour-suppressor genes, particularly deletions in , , , and . A subset of myxofibrosarcomas (MFS) was amplified for ( = 3) and ( = 1). was mutated in 7/15 cases of myxoid liposarcoma (MLS; 46.7%). Epigenetic regulators (e.g. and ) were frequently mutated.

Conclusions: In summary, FoundationOne Heme detected a broad range of genetic alterations and potential therapeutic targets in STS (e.g. in a subset of MFS, or in MLS). The assay's sensitivity for fusion detection was low in our sample and needs to be re-evaluated in a larger cohort.
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http://dx.doi.org/10.1177/17588359211029125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8317253PMC
July 2021

Expression of the cancer-associated DNA polymerase ε P286R in fission yeast leads to translesion synthesis polymerase dependent hypermutation and defective DNA replication.

PLoS Genet 2021 07 6;17(7):e1009526. Epub 2021 Jul 6.

ZRAB, University of Oxford, Oxford, United Kingdom.

Somatic and germline mutations in the proofreading domain of the replicative DNA polymerase ε (POLE-exonuclease domain mutations, POLE-EDMs) are frequently found in colorectal and endometrial cancers and, occasionally, in other tumours. POLE-associated cancers typically display hypermutation, and a unique mutational signature, with a predominance of C > A transversions in the context TCT and C > T transitions in the context TCG. To understand better the contribution of hypermutagenesis to tumour development, we have modelled the most recurrent POLE-EDM (POLE-P286R) in Schizosaccharomyces pombe. Whole-genome sequencing analysis revealed that the corresponding pol2-P287R allele also has a strong mutator effect in vivo, with a high frequency of base substitutions and relatively few indel mutations. The mutations are equally distributed across different genomic regions, but in the immediate vicinity there is an asymmetry in AT frequency. The most abundant base-pair changes are TCT > TAT transversions and, in contrast to human mutations, TCG > TTG transitions are not elevated, likely due to the absence of cytosine methylation in fission yeast. The pol2-P287R variant has an increased sensitivity to elevated dNTP levels and DNA damaging agents, and shows reduced viability on depletion of the Pfh1 helicase. In addition, S phase is aberrant and RPA foci are elevated, suggestive of ssDNA or DNA damage, and the pol2-P287R mutation is synthetically lethal with rad3 inactivation, indicative of checkpoint activation. Significantly, deletion of genes encoding some translesion synthesis polymerases, most notably Pol κ, partially suppresses pol2-P287R hypermutation, indicating that polymerase switching contributes to this phenotype.
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http://dx.doi.org/10.1371/journal.pgen.1009526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8284607PMC
July 2021

Cell-Free DNA Fragmentomics: The New "Omics" on the Block.

Clin Chem 2020 Dec;66(12):1480-1484

Technical Director, Diagnostic Molecular Pathology, Memorial Sloan Kettering Cancer Center, New York City, New York.

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http://dx.doi.org/10.1093/clinchem/hvaa258DOI Listing
December 2020

Vocal Fold Fibroblasts in Reinke's Edema Show Alterations Involved in Extracellular Matrix Production, Cytokine Response and Cell Cycle Control.

Biomedicines 2021 Jun 26;9(7). Epub 2021 Jun 26.

Division of Phoniatrics, Department of Otorhinolaryngology, Medical University of Graz, 8036 Graz, Austria.

The voice disorder Reinke's edema (RE) is a smoking- and voice-abuse associated benign lesion of the vocal folds, defined by an edema of the Reinke's space, accompanied by pathological microvasculature changes and immune cell infiltration. Vocal fold fibroblasts (VFF) are the main cell type of the lamina propria and play a key role in the disease progression. Current therapy is restricted to symptomatic treatment. Hence, there is an urgent need for a better understanding of the molecular causes of the disease. In the present study, we investigated differential expression profiles of RE and control VFF by means of RNA sequencing. In addition, fast gene set enrichment analysis (FGSEA) was performed in order to obtain involved biological processes, mRNA and protein levels of targets of interest were further evaluated. We identified 74 differentially regulated genes in total, 19 of which were upregulated and 55 downregulated. Differential expression analysis and FGSEA revealed upregulated genes and pathways involved in extracellular matrix (ECM) remodeling, inflammation and fibrosis. Downregulated genes and pathways were involved in ECM degradation, cell cycle control and proliferation. The current study addressed for the first time a direct comparison of VFF from RE to control and evaluated immediate functional consequences.
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http://dx.doi.org/10.3390/biomedicines9070735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8301432PMC
June 2021

Higher cMET dependence of sacral compared to clival chordoma cells: contributing to a better understanding of cMET in chordoma.

Sci Rep 2021 06 14;11(1):12466. Epub 2021 Jun 14.

Division of Biomedical Research, Medical University Graz, Graz, Austria.

Chordomas are rare slow growing, malignant bone tumors of the axial skeleton with no approved medical treatment. As the majority of chordomas express cMET and its ligand, HGF, and crosstalks between EGFR and MET-signaling exist, we aimed to explore cMET activity in chordoma cell lines and clinical samples. We investigated nine chordoma patients and four chordoma cell lines for cMET expression. Two clival and two sacral chordoma cell lines were tested for chromosomal abnormalities of the MET gene locus; we studied the influence of HGF on the autocrine secretion and migration behavior, as well as protein expression and phosphorylation. Two MET/ALK inhibitors were investigated for their effects on cell viability, cell cycle, cyclin alterations, apoptosis, and downstream signaling pathways. Moderate and strong expression of membrane and cytoplasmic cMET in chordoma patients and cell lines used, as well as concentration-dependent increase in phospho cMET expression after HGF stimulation in all four chordoma cell lines was shown. U-CH2, MUG-Chor1, and UM-Chor1 are polysomic for MET. Chordoma cell lines secreted EGF, VEGF, IL-6, and MMP9 upon HGF-stimulation. Sacral cell lines showed a distinct HGF-induced migration. Both inhibitors dose-dependently inhibited cell growth, induce apoptosis and cell-cycle arrest, and suppress downstream pathways. Heterogeneous responses obtained in our in vitro setting indicate that cMET inhibitors alone or in combination with other drugs might particularly benefit patients with sacral chordomas.
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http://dx.doi.org/10.1038/s41598-021-92018-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8203686PMC
June 2021

The clinical features of polymerase proof-reading associated polyposis (PPAP) and recommendations for patient management.

Fam Cancer 2021 May 5. Epub 2021 May 5.

Polyposis Registry, St Mark's Hospital, Harrow, London, HA1 3UJ, UK.

Pathogenic germline exonuclease domain (ED) variants of POLE and POLD1 cause the Mendelian dominant condition polymerase proof-reading associated polyposis (PPAP). We aimed to describe the clinical features of all PPAP patients with probably pathogenic variants. We identified patients with a variants mapping to the EDs of POLE or POLD1 from cancer genetics clinics, a colorectal cancer (CRC) clinical trial, and systematic review of the literature. We used multiple evidence sources to separate ED variants into those with strong evidence of pathogenicity and those of uncertain importance. We performed quantitative analysis of the risk of CRC, colorectal adenomas, endometrial cancer or any cancer in the former group. 132 individuals carried a probably pathogenic ED variant (105 POLE, 27 POLD1). The earliest malignancy was colorectal cancer at 14. The most common tumour types were colorectal, followed by endometrial in POLD1 heterozygotes and duodenal in POLE heterozygotes. POLD1-mutant cases were at a significantly higher risk of endometrial cancer than POLE heterozygotes. Five individuals with a POLE pathogenic variant, but none with a POLD1 pathogenic variant, developed ovarian cancer. Nine patients with POLE pathogenic variants and one with a POLD1 pathogenic variant developed brain tumours. Our data provide important evidence for PPAP management. Colonoscopic surveillance is recommended from age 14 and upper-gastrointestinal surveillance from age 25. The management of other tumour risks remains uncertain, but surveillance should be considered. In the absence of strong genotype-phenotype associations, these recommendations should apply to all PPAP patients.
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http://dx.doi.org/10.1007/s10689-021-00256-yDOI Listing
May 2021

Somatic Copy-Number Alterations in Plasma Circulating Tumor DNA from Advanced EGFR-Mutated Lung Adenocarcinoma Patients.

Biomolecules 2021 04 21;11(5). Epub 2021 Apr 21.

Comprehensive Cancer Center, Institute of Cancer Research, Department of Medicine I, Medical University of Vienna, 1090 Vienna, Austria.

Background: To assess the clinical relevance of genome-wide somatic copy-number alterations (SCNAs) in plasma circulating tumor DNA (ctDNA) from advanced epidermal growth factor receptor ()-mutated lung adenocarcinoma patients.

Methods: We included 43 patients with advanced T790M-positive lung adenocarcinoma who were treated with osimertinib after progression under previous EGFR-TKI therapy. We performed genomic profiling of ctDNA in plasma samples from each patient obtained pre-osimertinib and after patients developed resistance to osimertinib. SCNAs were detected by shallow whole-genome plasma sequencing and mutations were assessed by droplet digital PCR.

Results: SCNAs in resistance-related genes (rrSCNAs) were detected in 10 out of 31 (32%) evaluable patients before start of osimertinib. The presence of rrSCNAs in plasma before the initiation of osimertinib therapy was associated with a lower response rate to osimertinib (50% versus 81%, = 0.08) and was an independent predictor for shorter progression-free survival (adjusted HR 3.33, 95% CI 1.37-8.10, = 0.008) and overall survival (adjusted HR 2.54, 95% CI 1.09-5.92, = 0.03).

Conclusions: Genomic profiling of plasma ctDNA is clinically relevant and affects the efficacy and clinical outcome of osimertinib. Our approach enables the comprehensive assessment of SCNAs in plasma samples of lung adenocarcinoma patients and may help to guide genotype-specific therapeutic strategies in the future.
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http://dx.doi.org/10.3390/biom11050618DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8143372PMC
April 2021

Profiling of circulating tumor DNA and tumor tissue for treatment selection in patients with advanced and refractory carcinoma: a prospective, two-stage phase II Individualized Cancer Treatment trial.

Ther Adv Med Oncol 2021 27;13:1758835920987658. Epub 2021 Feb 27.

Division of Oncology, Department of Internal Medicine, Medical University of Graz, Graz, Austria.

Background: Molecular profiling (MP) represents an opportunity to match patients to a targeted therapy and when tumor tissue is unavailable, circulating tumor deoxyribonucleic acid (ctDNA) can be harnessed as a non-invasive analyte for this purpose. We evaluated the success of a targeted therapy selected by profiling of ctDNA and tissue in patients with advanced and refractory carcinoma.

Patients And Methods: A blood draw as well as an optional tissue biopsy were obtained for MP. Whole-genome sequencing and a cancer hotspot panel were performed, and publicly available databases were used to match the molecular profile to targeted treatments. The primary endpoint was the progression-free survival (PFS) ratio (PFS on MP-guided therapy/PFS on the last evidence-based therapy), whereas the success of the targeted therapy was defined as a PFS ratio ⩾1.2. To test the impact of molecular profile-treatment matching strategies, we retrospectively analyzed selected cases the CureMatch PreciGENE™ decision support algorithm.

Results: Interim analysis of 24 patients yielded informative results from 20 patients (83%). A potential tumor-specific drug could be matched in 11 patients (46%) and eight (33%) received a matched treatment. Median PFS in the matched treatment group was 61.5 days [interquartile range (IQR) 49.8-71.0] compared with 81.5 days (IQR 68.5-117.8) for the last evidence-based treatment, resulting in a median PFS ratio of 0.7 (IQR 0.6-0.9). Hence, as no patient experienced a PFS ratio ⩾1.2, the study was terminated. Except for one case, the CureMatch analysis identified either a two-drug or three-drug combination option.

Conclusions: Our study employed a histotype-agnostic approach to harness molecular profiling data from both ctDNA and metastatic tumor tissue. The outcome results indicate that more innovative approaches to study design and matching algorithms are necessary to achieve improved patient outcomes. EudraCT: 2014-005341-44.
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http://dx.doi.org/10.1177/1758835920987658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7923987PMC
February 2021

Detection of Aneuploidy in Cerebrospinal Fluid from Patients with Breast Cancer Can Improve Diagnosis of Leptomeningeal Metastases.

Clin Cancer Res 2021 May 29;27(10):2798-2806. Epub 2021 Jan 29.

Department of Medical Oncology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, the Netherlands.

Purpose: Detection of leptomeningeal metastasis is hampered by limited sensitivities of currently used techniques: MRI and cytology of cerebrospinal fluid (CSF). Detection of cell-free tumor DNA in CSF has been proposed as a tumor-specific candidate to detect leptomeningeal metastasis at an earlier stage. The aim of this study was to investigate mutation and aneuploidy status in CSF-derived cell-free DNA (cfDNA) of patients with breast cancer with a clinical suspicion of leptomeningeal metastasis.

Experimental Design: cfDNA was isolated from stored remnant CSF and analyzed by targeted next-generation sequencing (NGS; = 30) and the modified fast aneuploidy screening test-sequencing system (mFAST-SeqS; = 121). The latter method employs selective amplification of long interspaced nuclear elements sequences that are present throughout the genome and allow for fast and cheap detection of aneuploidy. We compared these results with the gold standard to diagnose leptomeningeal metastasis: cytology.

Results: Leptomeningeal metastasis was cytology proven in 13 of 121 patients. Low DNA yields resulted in insufficient molecular coverage of NGS for the majority of samples (success rate, 8/30). The mFAST-SeqS method, successful in 112 of 121 (93%) samples, detected genome-wide aneuploidy in 24 patients. Ten of these patients had cytology-proven leptomeningeal metastasis; 8 additional patients were either concurrently diagnosed with central nervous system metastases by radiological means or developed these soon after the lumbar puncture. The remaining six cases were suspected of leptomeningeal metastasis, but could not be confirmed by cytology or imaging. Aneuploidy was associated with development of leptomeningeal metastasis and significantly worse overall survival.

Conclusions: Aneuploidy in CSF-derived cfDNA may provide a promising biomarker to improve timely detection of leptomeningeal metastasis.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-3954DOI Listing
May 2021

On-treatment measurements of circulating tumor DNA during FOLFOX therapy in patients with colorectal cancer.

NPJ Precis Oncol 2020 Nov 13;4(1):30. Epub 2020 Nov 13.

Institute of Human Genetics, Diagnostic and Research Center for Molecular BioMedicine, Medical University of Graz, Graz, Austria.

We addressed a significant unknown feature of circulating tumor DNA (ctDNA), i.e., how ctDNA levels change during chemotherapy, by serially monitoring ctDNA in patients with colorectal cancer during the 48-h application of FOLFOX. Surprisingly, we did not observe a spike in ctDNA as a sign of a responsive tumor, but instead ctDNA levels initially decreased and remained low in patients with stable disease or partial response. Our observations reveal further insights into cell destruction during chemotherapy with important implications for the management of patients.
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http://dx.doi.org/10.1038/s41698-020-00134-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7666126PMC
November 2020

β-catenin regulates FOXP2 transcriptional activity via multiple binding sites.

FEBS J 2021 05 26;288(10):3261-3284. Epub 2020 Dec 26.

Gottfried Schatz Research Center for Cell Signaling, Metabolism and Aging, Molecular Biology and Biochemistry, Medical University of Graz, Austria.

The transcription factor forkhead box protein P2 (FOXP2) is a highly conserved key regulator of embryonal development. The molecular mechanisms of how FOXP2 regulates embryonal development, however, remain elusive. Using RNA sequencing, we identified the Wnt signaling pathway as key target of FOXP2-dependent transcriptional regulation. Using cell-based assays, we show that FOXP2 transcriptional activity is regulated by the Wnt coregulator β-catenin and that β-catenin contacts multiple regions within FOXP2. Using nuclear magnetic resonance spectroscopy, we uncovered the molecular details of these interactions. β-catenin contacts a disordered FOXP2 region with α-helical propensity via its folded armadillo domain, whereas the intrinsically disordered β-catenin N terminus and C terminus bind to the conserved FOXP2 DNA-binding domain. Using RNA sequencing, we confirmed that β-catenin indeed regulates transcriptional activity of FOXP2 and that the FOXP2 α-helical motif acts as a key regulatory element of FOXP2 transcriptional activity. Taken together, our findings provide first insight into novel regulatory interactions and help to understand the intricate mechanisms of FOXP2 function and (mis)-regulation in embryonal development and human diseases. DATABASE: Expression data are available in the GEO database under the accession number GSE138938.
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http://dx.doi.org/10.1111/febs.15656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8246981PMC
May 2021

Longitudinal tumor fraction trajectories predict risk of progression in metastatic HR breast cancer patients undergoing CDK4/6 treatment.

Mol Oncol 2021 Sep 18;15(9):2390-2400. Epub 2020 Dec 18.

Division of Oncology, Department of Internal Medicine, Medical University of Graz, Austria.

Despite improved clinical outcomes, intrinsic or acquired resistance to CDK4/6 inhibitor treatment has limited the success of this treatment in HR HER2 metastatic breast cancer patients. Biomarkers are urgently needed, and longitudinal biomarker measurements may harbor more dynamic predictive and prognostic information compared to single time point measurements. The aim of this study was to explore the longitudinal evolution of circulating tumor fractions within cell-free DNA assessed by an untargeted sequencing approach during CDK4/6 therapy and to quantify the potential association between longitudinal z-score measurements and clinical outcome by using joint models. Forty-nine HR HER2 metastatic breast cancer patients were enrolled, and z-score levels were measured at baseline and during 132 follow-up visits (median number of measurements per patient = 3, 25 -75 percentile: 3-5, range: 1-8). We observed higher baseline z-score levels (estimated difference 0.57, 95% CI: 0.147-0.983, P-value = 0.008) and a constant increase of z-score levels over follow-up time (overall P-value for difference in log z-score over time = 0.024) in patients who developed progressive disease. Importantly, the joint model revealed that elevated z-score trajectories were significantly associated with higher progression risk (HR of log z-score at any time of follow-up = 3.3, 95% CI, 1.44-7.55, P = 0.005). In contrast, single z-score measurement at CDK4/6 inhibitor treatment start did not predict risk of progression. In this prospective study, we demonstrate proof-of-concept that longitudinal z-score trajectories rather than single time point measurements may harbor important dynamic information on the development of disease progression in HR HER2 breast cancer patients undergoing CDK4/6 inhibitor treatment.
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http://dx.doi.org/10.1002/1878-0261.12870DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8410553PMC
September 2021

Comparison of three commercial decision support platforms for matching of next-generation sequencing results with therapies in patients with cancer.

ESMO Open 2020 09;5(5):e000872

Institute of Human Genetics, Diagnostic and Research Center for Molecular BioMedicine, Medical University of Graz, Graz, Austria; BioTechMed-Graz, Graz, Austria. Electronic address:

Objective: Precision oncology depends on translating molecular data into therapy recommendations. However, with the growing complexity of next-generation sequencing-based tests, clinical interpretation of somatic genomic mutations has evolved into a formidable task. Here, we compared the performance of three commercial clinical decision support tools, that is, NAVIFY Mutation Profiler (NAVIFY; Roche), QIAGEN Clinical Insight (QCI) Interpret (QIAGEN) and CureMatch Bionov (CureMatch).

Methods: In order to obtain the current status of the respective tumour genome, we analysed cell-free DNA from patients with metastatic breast, colorectal or non-small cell lung cancer. We evaluated somatic copy number alterations and in parallel applied a 77-gene panel (AVENIO ctDNA Expanded Panel). We then assessed the concordance of tier classification approaches between NAVIFY and QCI and compared the strategies to determine actionability among all three platforms. Finally, we quantified the alignment of treatment suggestions across all decision tools.

Results: Each platform varied in its mode of variant classification and strategy for identifying druggable targets and clinical trials, which resulted in major discrepancies. Even the frequency of concordant actionable events for tier I-A or tier I-B classifications was only 4.3%, 9.5% and 28.4% when comparing NAVIFY with QCI, NAVIFY with CureMatch and CureMatch with QCI, respectively, and the obtained treatment recommendations differed drastically.

Conclusions: Treatment decisions based on molecular markers appear at present to be arbitrary and dependent on the chosen strategy. As a consequence, tumours with identical molecular profiles would be differently treated, which challenges the promising concepts of genome-informed medicine.
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http://dx.doi.org/10.1136/esmoopen-2020-000872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7513637PMC
September 2020

A higher ctDNA fraction decreases survival in regorafenib-treated metastatic colorectal cancer patients. Results from the regorafenib's liquid biopsy translational biomarker phase II pilot study.

Int J Cancer 2021 03 16;148(6):1452-1461. Epub 2020 Oct 16.

Department of Medicine I, Division of Oncology, Medical University of Vienna, Vienna, Austria.

The predictive effect of circulating tumor DNA (ctDNA) in colorectal cancer (CRC) treatment is still highly discussed. The primary objective of our study was to investigate a possible prognostic/predictive value of ctDNA under regorafenib treatment. This prospective multicenter translational biomarker phase II pilot study enrolled 30 metastatic CRC patients (67% men, 33% women) treated with regorafenib. ctDNA was assessed in plasma before treatment start and at defined time points during administration. Measurement of tumor fraction as well as mutation and copy number analysis of CRC driver genes were performed by next-generation sequencing approaches. Multivariate analyses for survival and treatment efficacy were adjusted to age, gender and Eastern Cooperative Oncology Group. Disease control rate was 30%. Median tumor fraction at baseline was 18.5% (0-49.9). Mutations in CRC driver genes or genes involved in angiogenesis were identified in 25 patients (83.3%). KRAS mutations were detected in 13 of 14 KRAS-positive tumors; in three patients without KRAS mutation in the respective tumors, acquired mutations as a consequence of prior anti-EGFR treatment were detected. In a subset of patients, novel occurring mutations or focal amplifications were detected. A tumor fraction of 5% and higher at baseline was significantly associated with a decreased OS (P = .022; hazard ratio 3.110 (95% confidence interval: 1.2-8.2). ctDNA is detectable in a high proportion of mCRC patients. Higher ctDNA levels are associated with survival among regorafenib treatment. Moreover, our data highlight the benefit of a combined evaluation of mutations and somatic copy number alterations in advanced cancer patients.
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http://dx.doi.org/10.1002/ijc.33303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7894541PMC
March 2021

A Multi-Analyte Approach for Improved Sensitivity of Liquid Biopsies in Prostate Cancer.

Cancers (Basel) 2020 Aug 11;12(8). Epub 2020 Aug 11.

Division of Cell Biology, Histology and Embryology, Gottfried Schatz Research Center, Medical University of Graz, 8010 Graz, Austria.

Novel androgen receptor (AR) signaling inhibitors have improved the treatment of castration-resistant prostate cancer (CRPC). Nonetheless, the effect of these drugs is often time-limited and eventually most patients become resistant due to various AR alterations. Although liquid biopsy approaches are powerful tools for early detection of such therapy resistances, most assays investigate only a single resistance mechanism. In combination with the typically low abundance of circulating biomarkers, liquid biopsy assays are therefore informative only in a subset of patients. In this pilot study, we aimed to increase overall sensitivity for tumor-related information by combining three liquid biopsy approaches into a multi-analyte approach. In a cohort of 19 CRPC patients, we (1) enumerated and characterized circulating tumor cells (CTCs) by mRNA-based in situ padlock probe analysis, (2) used RT-qPCR to detect cancer-associated transcripts (e.g., and -splice variant 7) in lysed whole blood, and (3) conducted shallow whole-genome plasma sequencing to detect amplification. Although 44-53% of patient samples were informative for each assay, a combination of all three approaches led to improved diagnostic sensitivity, providing tumor-related information in 89% of patients. Additionally, distinct resistance mechanisms co-occurred in two patients, further reinforcing the implementation of multi-analyte liquid biopsy approaches.
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http://dx.doi.org/10.3390/cancers12082247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7465186PMC
August 2020

Sensitive and broadly applicable residual disease detection in acute myeloid leukemia using flow cytometry-based leukemic cell enrichment followed by mutational profiling.

Am J Hematol 2020 Jun 29. Epub 2020 Jun 29.

Division of Hematology, Medical University of Graz, Graz, Austria.

Persistent measurable residual disease (MRD) is an increasingly important prognostic marker in acute myeloid leukemia (AML). Currently, MRD is determined by multi-parameter flow cytometry (MFC) or PCR-based methods detecting leukemia-specific fusion transcripts and mutations. However, while MFC is highly operator-dependent and difficult to standardize, PCR-based methods are only available for a minority of AML patients. Here we describe a novel, highly sensitive and broadly applicable method for MRD detection by combining MFC-based leukemic cell enrichment using an optimized combinatorial antibody panel targeting CLL-1, TIM-3, CD123 and CD117, followed by mutational analysis of recurrently mutated genes in AML. In dilution experiments this method showed a sensitivity of 10 to 10 for residual disease detection. In prospectively collected remission samples this marker combination allowed for a median 67-fold cell enrichment with sufficient DNA quality for mutational analysis using next generation sequencing (NGS) or digital PCR in 39 out of 41 patients. Twenty-one samples (53.8%) tested MRD positive, whereas 18 (46.2%) were negative. With a median follow-up of 559 days, 71.4% of MRD positive (15/21) and 27.8% (5/18) of MRD negative patients relapsed (P = .007). The cumulative incidence of relapse (CIR) was higher for MRD positive patients (5-year CIR: 90.5% vs 28%, P < .001). In multivariate analysis, MRD positivity was a prominent factor for CIR. Thus, MFC-based leukemic cell enrichment using antibodies against CLL-1, TIM-3, CD123 and CD117 followed by mutational analysis allows high sensitive MRD detection and is informative on relapse risk in the majority of AML patients.
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http://dx.doi.org/10.1002/ajh.25918DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7540028PMC
June 2020

Technical Evaluation of Commercial Mutation Analysis Platforms and Reference Materials for Liquid Biopsy Profiling.

Cancers (Basel) 2020 Jun 16;12(6). Epub 2020 Jun 16.

Institute of Human Genetics, Diagnostic & Research Center for Molecular BioMedicine, Medical University of Graz, 8010 Graz, Austria.

Molecular profiling from liquid biopsy, in particular cell-free DNA (cfDNA), represents an attractive alternative to tissue biopsies for the detection of actionable targets and tumor monitoring. In addition to PCR-based assays, Next Generation Sequencing (NGS)-based cfDNA assays are now commercially available and are being increasingly adopted in clinical practice. However, the validity of these products as well as the clinical utility of cfDNA in the management of patients with cancer has yet to be proven. Within framework of the Innovative Medicines Initiative (IMI) program CANCER-ID we evaluated the use of commercially available reference materials designed for ctDNA testing and cfDNA derived from Diagnostic Leukaphereses (DLA) for inter- and intra-assay as well as intra- and inter-laboratory comparisons. In three experimental setups, a broad range of assays including ddPCR, MassARRAY and various NGS-based assays were tested. We demonstrate that both reference materials with predetermined VAFs and DLA samples are extremely useful for the performance assessment of mutation analysis platforms. Moreover, our data indicate a substantial variability of NGS assays with respect to sensitivity and specificity highlighting the importance of extensive validation of the test performance before offering these tests in clinical routine practice.
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http://dx.doi.org/10.3390/cancers12061588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352370PMC
June 2020

Shallow Whole-Genome Sequencing from Plasma Identifies FGFR1 Amplified Breast Cancers and Predicts Overall Survival.

Cancers (Basel) 2020 Jun 6;12(6). Epub 2020 Jun 6.

Division of Biotechnology, Servier Research Institute, 125, Chemin de ronde, 78290 Croissy Sur-seine, France.

Focal amplification of fibroblast growth factor receptor 1 () defines a subgroup of breast cancers with poor prognosis and high risk of recurrence. We sought to demonstrate the potential of circulating cell-free DNA (cfDNA) analysis to evaluate copy numbers from a cohort of 100 metastatic breast cancer (mBC) patients. Formalin-fixed paraffin-embedded (FFPE) tissue samples were screened for amplification by FISH, and positive cases were confirmed with a microarray platform (Oncoscan). Subsequently, cfDNA was evaluated by two approaches, i.e., mFAST-SeqS and shallow whole-genome sequencing (sWGS), to estimate the circulating tumor DNA (ctDNA) allele fraction (AF) and to evaluate the status. Tissue-based analyses identified amplifications in 20/100 tumors. All cases with a ctDNA AF above 3% ( = 12) showed concordance for status between tissue and cfDNA. In one case, we were able to detect a high-level amplification, although the ctDNA AF was below 1%. Furthermore, high levels of ctDNA indicated an association with unfavorable prognosis based on overall survival. Screening for amplification in ctDNA might represent a viable strategy to identify patients eligible for treatment by FGFR inhibition, and mBC ctDNA levels might be used for the evaluation of prognosis in clinical drug trials.
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http://dx.doi.org/10.3390/cancers12061481DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7353062PMC
June 2020

Assessment of Pre-Analytical Sample Handling Conditions for Comprehensive Liquid Biopsy Analysis.

J Mol Diagn 2020 08 1;22(8):1070-1086. Epub 2020 Jun 1.

Children's Cancer Research Institute, St. Anna Kinderkrebsforschung, Vienna, Austria; Department of Pediatrics, Medical University of Vienna, Vienna, Austria. Electronic address:

Liquid biopsies as a minimally invasive approach have the potential to revolutionize molecular diagnostics. Yet, although protocols for sample handling and the isolation of circulating tumor DNA (ctDNA) are numerous, comprehensive guidelines for diagnostics and research considering all aspects of real-life multicenter clinical studies are currently not available. These include limitations in sample volume, transport, and blood collection tubes. We tested the impact of commonly used (EDTA and heparin) and specialized blood collection tubes and storage conditions on the yield and purity of cell-free DNA for the application in down-stream analysis. Moreover, we evaluated the feasibility of a combined workflow for ctDNA and tumor cell genomic testing and parallel flow cytometric analysis of leukocytes. For genomic analyses, EDTA tubes showed good results if stored for a maximum of 4 hours at room temperature or for up to 24 hours when stored at 4°C. Spike-in experiments revealed that EDTA tubes in combination with density gradient centrifugation allowed the parallel isolation of ctDNA, leukocytes, and low amounts of tumor cells (0.1%) and their immunophenotyping by flow cytometry and down-stream genomic analysis by whole genome sequencing. In conclusion, adhering to time and temperature limits allows the use of routine EDTA blood samples for liquid biopsy analyses. We further provide a workflow enabling the parallel analysis of cell-free and cellular features for disease monitoring and for clonal evolution studies.
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http://dx.doi.org/10.1016/j.jmoldx.2020.05.006DOI Listing
August 2020

Cell-Free DNA and Apoptosis: How Dead Cells Inform About the Living.

Trends Mol Med 2020 05 17;26(5):519-528. Epub 2020 Feb 17.

Institute of Human Genetics, Diagnostic and Research Center for Molecular BioMedicine, Medical University of Graz, Graz, Austria; BioTechMed-Graz, Graz, Austria. Electronic address:

Cell-free DNA (cfDNA) is evolving into a widely used prognostic and predictive biomarker, particularly in oncology. However, its versatile clinical use precedes a profound understanding of the underlying biology of cfDNA release. There is much evidence to suggest that cfDNA is mainly derived from dying (i.e., apoptotic) cells. However, numerous cancer studies have shown that cfDNA is informative about acquired resistance to given therapies, which is present in living, proliferating tumor subclones. To explain this contradiction, we review current insights regarding cfDNA release, in particular the interplay between apoptosis and proliferation. We describe how improved knowledge about cfDNA biology could be used for novel therapeutic strategies and how this may affect patient management.
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http://dx.doi.org/10.1016/j.molmed.2020.01.012DOI Listing
May 2020

Publisher Correction: Inference of transcription factor binding from cell-free DNA enables tumor subtype prediction and early detection.

Nat Commun 2020 04 20;11(1):1965. Epub 2020 Apr 20.

Institute of Human Genetics, Diagnostic and Research Center for Molecular BioMedicine, Medical University of Graz, Graz, Austria.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-020-15799-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7170910PMC
April 2020

Functional Classification of Mutations in Acute Myeloid Leukemia.

Cancers (Basel) 2020 Mar 10;12(3). Epub 2020 Mar 10.

Division of Hematology, Medical University of Graz, A-8036 Graz, Austria.

Mutations of the gene occur in a subset of patients with acute myeloid leukemia (AML) and confer an exceedingly adverse prognosis. However, whether different types of mutations exert a uniformly poor outcome has not been investigated yet. Here, we addressed this issue by analyzing data of 1537 patients intensively treated within protocols of the German-Austrian AML study group. We classified mutations depending on their impact on protein structure and according to the evolutionary action (EAp53) score and the relative fitness score (RFS). In 98/1537 (6.4%) patients, 108 mutations were detected. While the discrimination depending on the protein structure and the EAp53 score did not show a survival difference, patients with low-risk and high-risk AML-specific RFS showed a different overall survival (OS; median, 12.9 versus 5.5 months, = 0.017) and event-free survival (EFS; median, 7.3 versus 5.2 months, = 0.054). In multivariable analyses adjusting for age, gender, white blood cell count, cytogenetic risk, type of AML, and TP53 variant allele frequency, these differences were statistically significant for both OS (HR, 2.14; 95% CI, 1.15-4.0; = 0.017) and EFS (HR, 1.97; 95% CI, 1.06-3.69; = 0.033). We conclude that the AML-specific RFS is of prognostic value in patients with TP53-mutated AML and a useful tool for therapeutic decision-making.
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http://dx.doi.org/10.3390/cancers12030637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7139772PMC
March 2020

Comprehensive characterization of cell-free tumor DNA in plasma and urine of patients with renal tumors.

Genome Med 2020 02 28;12(1):23. Epub 2020 Feb 28.

Hutchison/MRC Research Centre, University of Cambridge, Cambridge, CB2 0QQ, UK.

Background: Cell-free tumor-derived DNA (ctDNA) allows non-invasive monitoring of cancers, but its utility in renal cell cancer (RCC) has not been established.

Methods: Here, a combination of untargeted and targeted sequencing methods, applied to two independent cohorts of patients (n = 91) with various renal tumor subtypes, were used to determine ctDNA content in plasma and urine.

Results: Our data revealed lower plasma ctDNA levels in RCC relative to other cancers of similar size and stage, with untargeted detection in 27.5% of patients from both cohorts. A sensitive personalized approach, applied to plasma and urine from select patients (n = 22) improved detection to ~ 50%, including in patients with early-stage disease and even benign lesions. Detection in plasma, but not urine, was more frequent amongst patients with larger tumors and in those patients with venous tumor thrombus. With data from one extensively characterized patient, we observed that plasma and, for the first time, urine ctDNA may better represent tumor heterogeneity than a single tissue biopsy. Furthermore, in a subset of patients (n = 16), longitudinal sampling revealed that ctDNA can track disease course and may pre-empt radiological identification of minimal residual disease or disease progression on systemic therapy. Additional datasets will be required to validate these findings.

Conclusions: These data highlight RCC as a ctDNA-low malignancy. The biological reasons for this are yet to be determined. Nonetheless, our findings indicate potential clinical utility in the management of patients with renal tumors, provided improvement in isolation and detection approaches.
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http://dx.doi.org/10.1186/s13073-020-00723-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7048087PMC
February 2020

Cell-free DNA analysis reveals POLR1D-mediated resistance to bevacizumab in colorectal cancer.

Genome Med 2020 02 22;12(1):20. Epub 2020 Feb 22.

Institute of Human Genetics, Diagnostic and Research Center for Molecular Biomedicine, Medical University of Graz, Graz, Austria.

Background: Bevacizumab, a monoclonal antibody against soluble VEGFA, is an approved and commonly administered anti-angiogenic drug in patients with metastasized colorectal cancer (mCRC). The survival benefit of anti-VEGF therapy in mCRC patients is limited to a few months, and acquired resistance mechanisms are largely unknown. Here, we employed whole-genome sequencing of plasma DNA to evaluate the tumor genome of patients undergoing treatment with bevacizumab to determine novel aberrations associated with resistance.

Methods: Using longitudinal plasma analyses, we studied the evolution of tumor genomes in a mCRC cohort (n = 150) and conducted analyses of CRC cases from The Cancer Genome Atlas (TCGA) database (n = 619) to identify associations between genomic aberrations and clinical features. We employed whole-genome sequencing to identify the most frequently occurring focal somatic copy number alterations (SCNAs). Using the TCGA data as a comparative and supporting dataset, we defined the minimally amplified overlapping region and studied the mechanistic consequences of copy number gain of the involved genes in this segment. In addition, we established an in vitro cell model and conducted downstream gene expression and cell viability assays to confirm our findings from the patient dataset.

Results: We observed a recurrent focal amplification (8.7% of cases) on chromosome 13q12.2. Analysis of CRC cases from the TCGA database suggested that this amplicon is associated with more advanced stages. We confirmed that this 13q12.2 amplicon frequently emerges later during the clinical course of disease. After defining the minimally amplified region, we observed that the amplification and expression of one gene, POLR1D, impacted cell proliferation and resulted in upregulation of VEGFA, an important regulator of angiogenesis which has been implicated in the resistance to bevacizumab treatment. In fact, in several patients, we observed the emergence of this 13q12.2 amplicon under bevacizumab treatment, which was invariably associated with therapy resistance.

Conclusions: Non-invasive analyses of cell-free DNA from patients undergoing treatment with bevacizumab enabled the tracking of evolving tumor genomes and helped identify a recurrent focal SCNA of clinical relevance. Here, we describe a novel resistance mechanism against a widely applied treatment in patients with mCRC which will impact the clinical management of patients.
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http://dx.doi.org/10.1186/s13073-020-0719-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7036260PMC
February 2020

TP53 mutated AML subclones exhibit engraftment in a humanized bone marrow ossicle mouse model.

Ann Hematol 2020 Mar 30;99(3):653-655. Epub 2020 Jan 30.

Division of Hematology, Department of Internal Medicine, Medical University of Graz, Auenbruggerplatz 38, 8036, Graz, Austria.

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http://dx.doi.org/10.1007/s00277-020-03920-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060155PMC
March 2020

Novel phenotypes observed in patients with -linked leukaemia/familial thrombocytopenia syndrome and a biallelic risk allele as leukaemogenic cofactor.

J Med Genet 2020 06 8;57(6):427-433. Epub 2019 Nov 8.

Division of Pediatric Hemato-Oncology, Department of Pediatrics and Adolescent Medicine, Medical University of Graz, Graz, Austria

The phenotypes of patients with the recently discovered, dominant, -linked leukaemia predisposition and familial thrombocytopenia syndrome are variable, and the exact mechanism of leukaemogenesis remains unclear. Here, we present novel clinical and laboratory phenotypes of seven individuals from three families with germline mutations and a refined genetic analysis of one child with additional high-hyperdiploid acute lymphoblastic leukaemia (HD-ALL), aiming to elucidate second oncogenic hits. Four individuals from two pedigrees harboured one novel or one previously described variant in the central domain of (c.592C>T, p.Gln198* or c.641C>T, p.Pro241Leu, respectively). Neutropenia was an accompanying feature in one of these families that also harboured a variant in (c.1098_1103dup, p.Ile366_Gly367dup), while in the other, an autism-spectrum disorder was observed. In the third family, the index patient suffered from HD-ALL and life-threatening pulmonary mucor mycosis, and had a positive family history of 'immune' thrombocytopenia. Genetic analyses revealed a novel heterozygous mutation in the ETS domain of (c.1136T>C, p.Leu379Pro) along with absence of heterozygosity of chromosome (10)(q21.2q21.3), yielding a biallelic leukaemia risk allele in (rs7090445-C). The neutrophil function was normal in all individuals tested, and the platelet immune histochemistry of all three pedigrees showed delta-storage-pool defect-like features and cytoskeletal defects. Our clinical observations and results of high-resolution genetic analyses extend the spectrum of possible phenotypes cosegregating with germline mutations. Further, we propose as potential leukaemogenic cofactor in patients with -linked leukaemia predisposition and familial thrombocytopenia syndrome.
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http://dx.doi.org/10.1136/jmedgenet-2019-106339DOI Listing
June 2020

Circulating Tumor DNA for Modern Cancer Management.

Authors:
Ellen Heitzer

Clin Chem 2019 Oct 31. Epub 2019 Oct 31.

Institute of Human Genetics, Christian Doppler Laboratory for Liquid Biopsies for Early Detection of Cancer, Diagnostic and Research Center for Molecular BioMedicine, Medical University of Graz, Graz, Austria.

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http://dx.doi.org/10.1373/clinchem.2019.304774DOI Listing
October 2019

Genome-Wide Analysis of the Nucleosome Landscape in Individuals with Coffin-Siris Syndrome.

Cytogenet Genome Res 2019 26;159(1):1-11. Epub 2019 Oct 26.

The switch/sucrose non-fermenting (SWI/SNF) complex is an ATP-dependent chromatin remodeller that regulates the spacing of nucleosomes and thereby controls gene expression. Heterozygous mutations in genes encoding subunits of the SWI/SNF complex have been reported in individuals with Coffin-Siris syndrome (CSS), with the majority of the mutations in ARID1B. CSS is a rare congenital disorder characterized by facial dysmorphisms, digital anomalies, and variable intellectual disability. We hypothesized that mutations in genes encoding subunits of the ubiquitously expressed SWI/SNF complex may lead to alterations of the nucleosome profiles in different cell types. We performed the first study on CSS-patient samples and investigated the nucleosome landscapes of cell-free DNA (cfDNA) isolated from blood plasma by whole-genome sequencing. In addition, we studied the nucleosome landscapes of CD14+ monocytes from CSS-affected individuals by nucleosome occupancy and methylome-sequencing (NOMe-seq) as well as their expression profiles. In cfDNA of CSS-affected individuals with heterozygous ARID1B mutations, we did not observe major changes in the nucleosome profile around transcription start sites. In CD14+ monocytes, we found few genomic regions with different nucleosome occupancy when compared to controls. RNA-seq analysis of CD14+ monocytes of these individuals detected only few differentially expressed genes, which were not in proximity to any of the identified differential nucleosome-depleted regions. In conclusion, we show that heterozygous mutations in the human SWI/SNF subunit ARID1B do not have a major impact on the nucleosome landscape or gene expression in blood cells. This might be due to functional redundancy, cell-type specificity, or alternative functions of ARID1B.
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http://dx.doi.org/10.1159/000503266DOI Listing
December 2019

Multicenter Evaluation of Circulating Cell-Free DNA Extraction and Downstream Analyses for the Development of Standardized (Pre)analytical Work Flows.

Clin Chem 2020 01;66(1):149-160

QIAGEN GmbH, Hilden, Germany.

Background: In cancer patients, circulating cell-free DNA (ccfDNA) can contain tumor-derived DNA (ctDNA), which enables noninvasive diagnosis, real-time monitoring, and treatment susceptibility testing. However, ctDNA fractions are highly variable, which challenges downstream applications. Therefore, established preanalytical work flows in combination with cost-efficient and reproducible reference materials for ccfDNA analyses are crucial for analytical validity and subsequently for clinical decision-making.

Methods: We describe the efforts of the Innovative Medicines Initiative consortium CANCER-ID (http://www.cancer-id.eu) for comparing different technologies for ccfDNA purification, quantification, and characterization in a multicenter setting. To this end, in-house generated mononucleosomal DNA (mnDNA) from lung cancer cell lines carrying known TP53 mutations was spiked in pools of plasma from healthy donors generated from 2 different blood collection tubes (BCTs). ccfDNA extraction was performed at 15 partner sites according to their respective routine practice. Downstream analysis of ccfDNA with respect to recovery, integrity, and mutation analysis was performed centralized at 4 different sites.

Results: We demonstrate suitability of mnDNA as a surrogate for ccfDNA as a process quality control from nucleic acid extraction to mutation detection. Although automated extraction protocols and quantitative PCR-based quantification methods yielded the most consistent and precise results, some kits preferentially recovered spiked mnDNA over endogenous ccfDNA. Mutated TP53 fragments derived from mnDNA were consistently detected using both next-generation sequencing-based deep sequencing and droplet digital PCR independently of BCT.

Conclusions: This comprehensive multicenter comparison of ccfDNA preanalytical and analytical work flows is an important contribution to establishing evidence-based guidelines for clinically feasible (pre)analytical work flows.
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http://dx.doi.org/10.1373/clinchem.2019.306837DOI Listing
January 2020
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